Plants perceive light in the environment using a number of photoreceptor systems. Phytochrome is the best characterized of these photoreceptors, having been shown to play a critical role in regulating growth and development throughout the life cycle of the plant. It has been shown to be functionally involved in the control of a wide range of biological and developmental functions in plants, including such processes as de-etiolation of germinating seedlings, regulation of the synthesis of a host of plastid proteins such as those of the photosynthetic apparatus, control of shade tolerance, and regulation of the timing of flowering and fruit production [for review, see Shropshire, W., Jr., and Mohr, H. (eds.) "Photomorphorphogenesis", Encyclopedia of Plant Physiology, New Series, Vols. 16A and 16B. Springer verlag; Heidelberg/Berlin 1983]. The phytochrome molecule is now believed to be a homodimer of subunits, each known to consist of a linear tetrapyrrole chromophore covalently attached to a polypeptide backbone via a thioether linkage. The size of the polypeptide portion of the photoreceptor varies from 120-127 kilodaltons among p)ant species [Vierstra et al., Planta. 160: 521-528 (1984)]. Phytochrome exists in plants in two spectrally distinct forms; the Pr form that absorbs maximally in the red (.lambda.max=666 nm) region of the spectrum and the Pfr form that absorbs maximally in the far-red (.lambda.max=730 nm) region of the spectrum. The two forms are reversibly interconvertible by light; Pr is converted to Pfr by absorbing red light and Pfr is converted to Pr by absorbing far-red light. In vivo, photoconversion of Pr to Pfr by red light induces a vast array of morphogenic responses whereas reconversion of Pfr back to Pr by far-red light cancels the induction of these responses. It is this property of indefinitely repeatable photointerconvertibility that allows phytochrome to function essentially as a reversible regulatory switch, with Pr and Pfr considered to be the biologically inactive and active forms of the molecule, respectively. For a general discussion of the photoconversion process, see Moses and Chua [Moses, P. B. and Chua, N. H., Sci. Amer., 258(4):88-93 (1988)].
Although the pathway from light perception by the photoreceptor to changes in the transcription pattern of nuclear genes remains unknown, the function of phytochrome is well defined in terms of its regulation of a vast number of biological processes. Consequently, it may be that altering phytochrome levels in light-grown plants will provide a unique opportunity to noninvasively influence the complex developmental systems that regulate plant development. Further, since phytochrome plays such an important regulatory role throughout the life cycle of plants, modification of steady state phytochrome concentrations in the cells of light-grown plants may result in the creation of a number of desirable growth and developmental traits that could have potential agronomic benefit. These benefits may be in the form of genetically engineered plants with altered phenotypes such that these new phenotypes provide the plants with advantages in the field compared with their normal counterparts, thus giving rise to new plant varieties possessing improved agronomic value.
In addition, the phytochrome system is known to interact in a yet unknown way with the processes that regulate phytohormone balances in plant tissues. Modification of steady-state phytochrome levels in transgenic plants may change the balances between the various endogenous phytohormones that govern both plant morphology and development. Alterations in the steady state phytochrome levels in the tissues of light-grown plants may have dramatic effects on the development and morphology of modified plants through disruption of normal phytohormone balances, and thereby potentially give rise to plants with new traits that have agronomic value.
The genetic modification of plants to create new and useful phenotypes by altering phytochrome levels in light-grown plants through modification of plant growth and morphology is a novel concept. Hershey et al. [Hershey, H. P., Barker, R. F., Idler, K. B., Murray, M. G., and Quail, P. H., Gene 61: 339-348, (1987)] have reported the first elucidation of the sequence and organization of a phytochrome gene from a plant. The nucleotide sequence of the gene is presented along with its intron/exon organization determined from a comparison of the gene sequence with sequences derived from the corresponding cDNA clones. In addition, data is given showing the transcription start site of the gene. Sequence elements in the 5' flanking region of the phytochrome gene that may participate in its light-regulation are suggested based upon the homology of these elements with other light-regulated genes. The authors discuss the possibility of expressing phytochrome with its native promoter in a transgenic system as a method to study the role of the proposed promoter elements in the light-regulation of phytochrome gene transcription. There is no disclosure of the value or impact of either the expression or the overexpression of phytochrome on the phenotype of transformed plants.
Nagy et al. [Nagy, F., Kay, S. A., and Chua, N. H., Trends in Genetics. 4: 37-42 (1988)] review the state of phytochrome research with particular emphasis on elucidation of the signal transduction pathway that leads from the red-light induced photoconversion of Pr to Pfr through to the changes in the expression of genes regulated by phytochrome. As background for the discussion on signal transduction, the authors discuss properties of the photoreceptor, its protein structure, its localization in the plant and within the cell, its different forms, and the status of efforts to clone phytochrome coding sequences. The nature of the transcriptional regulation of phytochrome-controlled genes is discussed with emphasis on DNA sequences and trans-acting proteins which participate in this regulation. Models for gene regulation and for signal transduction are proposed.
Nagy et al. (cited above) discuss future directions to be taken in phytochrome research with particular emphasis on elucidation of the signal transduction pathway. The testing of phytochrome genes which have been subjected to site directed mutagenesis in transgenic plants is suggested as a way to better understand structure/function relationships between phytochrome protein structure and altered gene expression. In this context, the authors propose overproduction of phytochrome and/or expression of the photoreceptor in inappropriate cell types as a tool to study how these changes in expression affect other phytochrome-controlled genes. They also propose the creation of dominant mutations in the phytochrome protein in order to study the signal transduction pathway by altering the functional structure of the photoreceptor. The authors concentrate their discussion and speculations on regulation of gene expression by the photoreceptor rather than on what affect, if any, expression of phytochrome would have either on gene expression or on whole plant responses in transgenic plants. In addition, no discussion of potential agronomic benefit that might result from phytochrome overexpression in whole plants appears in the article. Finally, no reports of attempts to express phytochrome in transgenic plants, either on its native promoter or by fusing a phytochrome coding sequence to a highly active constitutive promoter are disclosed in the Nagy et al. article (cited above).
Another recent review discusses the current understanding of light regulation of nuclear and chloroplast gene expression by phytochrome and UV light photoreceptors. [Jordan, B. Thomas, and Partis, M.D., in 1986 BCPC Mono No. 34 Biotechnology and Crop Improvement and Protection. Pp. 49-59, (1986)]. Suggestions are made for methods by which light-regulated genes can be manipulated to improve crop productivity. These include 1) mutational alteration of the RUBP carboxylase molecule and photoinactivation of other enzymes involved in photorespiration; 2) genetic engineering of the thylakoid membrane proteins to confer herbicide resistance and increase their abundance under low light conditions; 3) the use of controlling (promoter) sequences from light-regulated genes to control genes which are not normally light-regulated; and 4) the manipulation of the photoperiodic regulation of flowering and seed production to give more control of these processes. No specific approach to manipulating photoperiod is given and no mention is made of the possibility of genetically engineering any known plant photoreceptor to modify crop productivity.
The only known reports that discuss plants with changes in their phytochrome levels relate to mutants of tomato and arabidopsis that appear to have reduced levels of spectrally detectable phytochrome as compared to wild type plants. The mutants have been reported to display some characteristics that are somewhat similar to those seen in etiolated plants, i.e. they have more elongated hypocotyls, are more yellowish than their normal healthy green, counterparts, and appear to display some increased apical dominance [Koorneeff, M., Cone, J. W., Dekens, R. G., O'Herne-Robers, E. G., Sproit, C., J. P. and Kendricle, R. E., J. Plant Physiol. 120:153-165 (1985)]. No data relating to the effect of the mutation on yield or any other trait of potential agronomic value was presented, and only mutational reduction in the level of the Photoreceptor was considered.
To date, there is only one report of the isolation of a DNA fragment that contains an uninterrupted coding sequence for a complete phytochrome polypeptide [Lissemore, J. L., Colbert, J. T., Quail, P. H., Plant Molecular Biology, 8: 485-496 (1987)]. This article discloses the isolation of a full length cDNA clone for phytochrome from Curcurbita pepo (zucchini). However, unlike phytochrome gene expression in Avena (oats), the expression of the endogenous Curcurbita phytochrome gene is under weak, if any, phytochrome control since it displayed almost no response to Pr/Pfr photoconversion. Since the investigators did not test the responsiveness in zucchini of other genes known to be under phytochrome control in other plant species, it remains unknown if the lack of responsiveness of the zucchini phytochrome genes are due simply to a difference in the light-responsiveness of the gene or if there is some fundamental difference in regulatory function between Curcurbita and Avena phytochromes at the level of nuclear gene expression. In the absence of any further data, it seems prudent that attempts to overexpress phytochrome in transgenic plants should be made with an Avena type coding sequence since it encodes a polypeptide that is known to exert strong regulatory influence over nuclear gene expression in its native species.
Various DNA fragments containing partial Avena coding regions are available which can be combined to provide the necessary coding information to synthesize a complete phytochrome polypeptide. However, while Avena genomic clones can be combined to make a single DNA fragment encoding a complete photoreceptor molecule, the coding region would be dispersed among a number of exons as all known Avena genes contain introns. This makes existing genomic DNA fragments poor starting materials for creation of a coding region that is generally useful for expression in any transgenic plant since there is recent evidence that the introns of genes from monocotyledonous plants (of which Avena is an example) are processed poorly in dicotyledonous species. [Keith, B., and Chua, N. H., EMBO J., 5:2419-2425 (1986)]. It is, therefore, necessary to produce an uninterrupted Avena phytochrome coding sequence for expression in transgenic plants since no clones containing a generally useful protein coding region are available.
Since it is well documented that light-induced differences in the conformational structures of Pr and Pfr are fundamentally involved in the mechanism of action of the photoreceptor, any changes in amino acid composition of phytochrome may negatively affect the biological efficacy of the altered protein. While it is known that the protein domains that are involved in dimerization of the subunits of phytochrome are found in the C-terminal 30 kilodaltons of the protein [Vierstra et al., 1984, as described above, Jones and Quail, Biochemistry, 25:2987-2995 (1986)] and the domains responsible for Pr/Pfr photoconversion are in the N-terminal 70 kilodaltons [Vierstra et al., 1984, as above, Wong et al., J. Biol. Chem., 261:12089-12097 (1986)] the precise amino acids involved in these functions as well as those involved in adoption and maintenance of the Pr and Pfr secondary and higher order structures of the dimer have yet to be elucidated. Therefore, since the new phytochrome polypeptide used in this work differs from the type 3 phytochrome at 5 amino acids and differs from the type 4 phytochrome at 20 amino acids, it was unknown if the protein would be active since any single amino acid change or combination of changes could adversely affect the three dimensional structure of the protein and thus its biological activity. Indeed, it was even unknown if a monocotyledonous phytochrome would function in dicotyledonous species since there is only 65% protein homology between the only known monocot (Avena) and dicot (Cucurbita) phytochrome sequences [Sharrock et al., Gene, 47:287-295 (1986)].
Despite considerable research on the phytochrome peptide, a coding region has not been isolated which has been shown to be generally functional in both monocotyledonous and dicotyledonous plants. Furthermore, transformation constructs do not exist for overexpression of phytochrome in any plant species. The agronomic benefits of overexpression of phytochrome have therefore not been achieved.